Rheumatoid arthritis (RA) is an autoimmune synovitis that affects 0.5% of the world population, yet the key autoantigen targets remain unknown. Production of autoantibodies, such as the anti-citrullinated protein antibodies (ACPAs), is a hallmark of RA. However, which antigens the critical ACPAs and other RA- associated autoantibodies target remains largely unknown. Also unknown is how these ACPAs develop, how similar the ACPA repertoires are between different individuals with RA, and whether and how they contribute to the pathogenesis of RA. So far, the research field has lacked the means to comprehensively characterize the autoantibodies associated with a given disease and to then rationally winnow them to those that are important-that is, those that either drive the disease or serve as identifiers of the key antigens that trigger the pathogenic T-cell response. We have now developed antibody repertoire capture technology, a high- throughput method that allows us to do just that. Harnessing the power of next-generation sequencing, we have developed a novel method for barcoding all the cDNAs generated from individual antibody-expressing cells, thereby enabling high-throughput analysis of the paired heavy- and light-chain immunoglobulin genes expressed by single B cells, plasmablasts, or plasma cells. We hypothesize that we can elucidate the pathogenic autoantibody responses associated with RA by defining the antibody repertoire of plasmablasts and antigen-sorted memory B cells in the blood, and of plasmablasts and plasma cells in the synovium, of individuals with RA. We will then bioinformatically analyze the antibody sequences we obtain to generate evolutionary trees of the antibody repertoires and thereby identify and clone the affinity-matured antibodies that are likely the key autoantibodies. We will identify the antigens targeted by these recombinant, affinity-matured autoantibodies, investigate how their specific sequences develop, dissect their binding and immunostimulatory properties, and assess their pathogenicity. Success of this proposal would shed light on the development of the autoantibody response and identify the key autoantigens targeted in RA, findings that could lead to the development of new diagnostics and therapies for RA.